Determination of caffeine and its metabolites simultaneously in vivo by HPLC and used for enzyme assays on pharmacological study

A precise, accurate and validated HPLC method is developed for the determination of caffeine and its metabolites in vivo and used for enzyme assays. The separation is performed on a Hypersil BDS C18 column. The mobile phase composed by phase A (acetonitrile) and phase B (10mM ammonium formate/formic acid (998/2, v/v)) pumped according to a gradient elution program at a flow rate of 1ml·min-1 with detection at 280nm. Chloroform/isopropanol(9:1,v/v) is used as extractant, acetaminophen is selected as internal standard(IS). AFMU, 1U, 1X, IS, 17U, 17X and caffeine elute at 3.3, 7.3, 8.9, 10.7, 11.7, 12.1, 15.0 respectively, linear response (r>0.99) is observed for samples ranging from 1.25 to 160mM and the method provides recoveries of 81-94% in the concentration range of 1.25-80mM for these substances, intra- and inter-day variation in <10% R.S.D. The limit of detection (LOD) is 0.62mM for AFMU, 1X, 1U and 0.31mM for 17U, 17X, 137X.